Spectro-temporal optical encoding of information using a time-gated fluorescence resonance energy transfer (FRET)

ABSTRACT

Described herein is a time-gated, two-step FRET relay effective to provide temporal transference of a prompt FRET pathway, or provide spectro-temporal encoding analytical signals and other information. A FRET relay assembly includes a long lifetime FRET donor (for example, a lanthanide complex), a semiconductor quantum dot (QD) configured as an intermediate acceptor/donor in FRET, and a fluorescent dye configured as a terminal FRET acceptor, wherein the long lifetime FRET donor has an excited state lifetime of at least one microsecond and the QD and fluorescent dye each have excited state lifetimes of less than 100 nanoseconds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of as a divisional of U.S. patentapplication Ser. No. 13/475,177 filed on May 18, 2012, the entirety ofwhich is incorporated herein by reference.

BACKGROUND

The introduction of luminescent semiconductor nanocrystals or quantumdots (QDs) to biology has provided researchers with novel fluorescenttools for potentially achieving advances in imaging and sensing. See,for example, U.S. Patent Application Publication Nos. 2008/0087843 and2011/0089241, each of which is incorporated herein by reference. Inparticular, QDs have been widely adopted as either donors or acceptorsin Förster resonance energy transfer (FRET)-based assays and biosensors.

FRET has been used in a wide variety of applications, including: staticdistance measurements within or between (bio)molecules (i.e. FRET as a“spectroscopic ruler” between ˜1-10 nm); dynamic observation of changesin biomolecular conformation; diagnostic constructs that use changesbetween FRET “on” (high efficiency) and “off” (low efficiency) states todetect chemical/biological analytes; and light harvesting/photonicwires. In spectroscopic ruler contexts, FRET is generally implemented inits simplest configuration, which comprises a single donor luminophoreand single acceptor chromophore. The FRET efficiency can be used toderive the donor-acceptor separation distance and is almost alwaysmeasured on the basis of quenching of the donor luminescence intensityor decrease in the donor excited state lifetime. When the acceptorchromophore is also fluorescent, the ratio of acceptor and donorluminescence intensities can be a useful qualitative or quantitativemeasure. Diagnostic probes (for example, molecular beacons, Scorpionprimers, or TaqMan probes) also predominately utilize discretedonor-acceptor pairs. Multi-step FRET relays have been describedpreviously, using only prompt (nanosecond scale) fluorescence. Theprimary purpose has been to extend the net range of FRET or serve as aphotonic wire.

Described herein is a time-gated, two-step FRET relay effective toprovide temporal transference of a prompt FRET pathway, or providespectro-temporal encoding.

BRIEF SUMMARY

In one embodiment, a FRET relay assembly includes a long lifetime FRETdonor, a semiconductor quantum dot (QD) configured as an intermediateacceptor/donor in FRET, and a fluorescent dye configured as a terminalFRET acceptor, wherein the long lifetime FRET donor has an excited statelifetime of at least one microsecond and the QD and fluorescent dye eachhave excited state lifetimes of less than 100 nanoseconds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary embodiments illustrating the concept of thetime-gated, two-step FRET relay. FIG. 1A (i) shows time-gated FRETsensitization of QD photoluminescence (PL) via FRET₁. Both the Tb and QDare initially excited by a flash of UV light; however, the QD relaxes toits ground state after a suitable microsecond delay (time gate) andbecomes a good FRET acceptor for a proximal long-lifetime Tb donor. FIG.1A (ii) shows time-gated sensitization of A647 PL via FRET₁ and FRET₂.The co-assembly of a fluorescent dye, A647, with the Tb around a QDpermits a two-step energy transfer relay with the QD as an intermediary.In FIG. 1B, the QD is able to serve as a nanoscaffold for the controlledassembly of biomolecules labeled with Tb and A647.

FIG. 2A shows absorbance and PL spectra for the Tb³⁺ complex (with apeptide in a complex termed PEP B-Tb) (the compositions of this andother complexes are detailed below), red-emitting CdSe/ZnS core/shellQDs, and A647 dye (as PEP A-A647); 339 and 400 nm excitation are shownfor reference. FIG. 2B shows spectral overlap functions for the Tb-QDand QD-A647 FRET pairs.

FIG. 3 shows time-gated (with a 55 μs delay before data collection) PLspectra showing the increasing FRET₁-sensitization of QD PL with anincreasing amount of: PEP B-Tb (FIG. 3A) and PRB B/TGT B-Tb (FIG. 3B)(where “PRB” refers to a probe) hybrids (the valence of PRB B isconsistently 20). In each case, the insets show an approximately linearincrease in FRET₁-sensitized QD PL. FIG. 3C shows non-gated (˜0 μs) andtime-gated (55 μs) PL excitation spectra for QD, PEP B-Tb, andconjugates collected at Tb (490 nm) and QD (625 nm) PL emissionwavelengths.

FIG. 4 shows Tb donor PL decay curves collected at (A) 490 nm and (B)550 nm for different PEP B-Tb per QD ratios. (C) Higher-resolution donorPL decay curves collected at 490 nm for the indicated PEP B-Tb per QDratios. (D) Magnified view of the short PL lifetime component for thedata in (C). The small fraction of short PL lifetime component in thegreen (Tb only) curve is an artifact or echo from the detection setup.(E) Native QD PL decay curves. (F) FRET₁-sensitized QD PL decay curvesat different PEP B-Tb per QD ratios. The QD reflects the ˜10² μslifetime associated with energy transfer from the Tb.

FIG. 5A shows non-gated (˜0 μs) and time-gated (55 μs) PL spectra forQD-(PEP A-A647), assemblies with increasing A647 ratio or valence (m).No PL was observed in the time-gated spectrum. FIG. 5B shows PL spectraof (PEP B-Tb)₁₀-QD-(PEP A-A647), assemblies. QD and A647 PL are apparentin both the non-gated and time-gated spectra. FIG. 5C shows correlationof the FRET efficiencies as a function of A647 valence between the threedifferent sets of PL spectra in (A) and (B), calculated from the degreeof QD donor quenching. The inset shows the corresponding A647/QD PLratios. FIG. 5D shows A647 PL excitation spectra for the differentconfigurations, illustrating both FRET₁ and FRET₂ sensitization.

FIG. 6 shows time-gated biosensing configurations with a two-stepQD-FRET relay. FIG. 6A shows times courses of trypsin proteolyticactivity using (PEP B-Tb)₁₀-QD-(PEP A-A647)₃ assemblies. The time-gated(i) QD and (ii) A647 PL were monitored and converted to (iii) FRETefficiency. Dashed lines represent tangents drawn to calculate theinitial rate. (iv) The initial rate of change of FRET efficiency wasproportional to the trypsin concentration. FIG. 6B(i) shows PL spectrafor the non-gated calibration of TGT A-A647 hybridization using QD-(PRBA)₁₅ assemblies. The inset shows the FRET efficiency and A647/QD PLratio as a function of TGT A-A647 per QD. FIG. 6B(ii) shows time-gatedsensing of TGT A-A647 using (PEP B-Tb)₁₀-QD-(PRB A)₁₂ assemblies. Theinset shows the FRET efficiency and A647/QD PL ratio as a function ofTGT A-A647 concentration. Note: the corresponding TGT A-A647 added isgiven in equivalents in (i) and concentration in (ii).

FIG. 7 shows time-gated two-plex sensing of nucleic acid hybridizationusing a two-step QD-FRET relay. (A) PL spectra showing thecharacteristic non-gated (˜0 μs) and time-gated (55 μs) response of (PRBB)₁₆-QD-(PRB A)₁₀ assemblies: (i) no target; (ii) TGT A-A647; (iii) TGTB-Tb; and (iv) TGT A-A647 and TGT B-Tb. The solid black lines showscaling of the non-gated PL spectrum to fit the time-gated PL spectrum(via numerical deconvolution). (B) Orthogonal calibrations curves basedon measurement of: (i) the non-gated A647/QD PL ratio, and (ii)time-gated total QD+A647 PL sensitization. Each parameter respondedlinearly to increasing amounts of the corresponding target, and wasapproximately independent of the other analytical parameter.

FIG. 8 illustrates exemplary bioconjugate chemistries. (A) Pyridyldisulfide activation of thiolated-peptide and a disulfide exchangereaction to prepare His₆-peptide-oligonucleotide chimeras. (B)A647-maleimide PEP A labeling at an N-terminal cysteine residue. (C)Tb-NHS PEB B labeling at the N-terminus. (D) Tb-NHS labeling at an aminomodified linker of TGT B. Actual linker structures shown for the amineand thiol reactions. (E) Lumi4® NHS ligand structure (Tb³⁺ omitted forclarity)

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used in this specification and the appended claims, the singularforms “a”, “an,” and “the” do not preclude plural referents, unless thecontent clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

FRET refers to Fluorescence Resonance Energy Transfer, also termedFörster Resonance Energy Transfer.

The term “quantum dot” or “QD” as used herein refers to an inorganicsemiconductor crystallite of about 1 nm or more and about 1000 nm orless in diameter or any integer or fraction of an integer therebetween,preferably at least about 2 nm and about 50 nm or less in diameter orany integer or fraction of an integer therebetween, more preferably atleast about 2 nm and about 20 nm or less in diameter (for example about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nm). QDs are characterized by their substantially uniform nanometersize, frequently exhibiting approximately a 10% to 15% polydispersion orrange in size. A QD is capable of emitting electromagnetic radiationupon excitation (i.e., the QD is photoluminescent) and includes a “core”of one or more first semiconductor materials, and may be surrounded by a“shell” of a second semiconductor material. A QD core surrounded by asemiconductor shell is referred to as a “core/shell” QD. The surrounding“shell” material will preferably have a bandgap energy that is largerthan the bandgap energy of the core material and may be chosen to havean atomic spacing close to that of the “core” substrate.

The core and/or the shell can be a semiconductor material including, butnot limited to, those of the groups II-VI (ZnS, ZnSe, ZnTe, US, CdSe,CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe,SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb,InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like)materials, PbS, PbSe, and an alloy or a mixture thereof. Preferred shellmaterials include ZnS.

A QD is optionally surrounded by a “coat” of an organic capping agent.The organic capping agent may be any number of materials, but has anaffinity for the QD surface. The coat can be used to convey solubility,e.g., the ability to disperse a coated QD homogeneously into a chosensolvent, functionality, binding properties, or the like. In addition,the coat can be used to tailor the optical properties of the QD. Ingeneral, the capping agent can be an isolated organic molecule, apolymer (or a monomer for a polymerization reaction), an inorganiccomplex, or an extended crystalline or amorphous structure. The two mostcommon strategies for imparting aqueous solubility are the use ofbifunctional ligand coatings and bifunctional polymer coatings. Theformer coordinate to the QD surface through a chemical function (e.g.,thiol, imidazole) and replace the native hydrophobic ligands; the latterhave pendant alkyl chains that interdigitate with the native ligands viahydrophobic interactions. In both cases, a second and polar chemicalfunction—such as a carboxylate, amine, or poly(ethylene glycol) (PEG)chain—mediates aqueous solubility. Mixtures of different ligands and theuse of copolymers enable the modification of QDs with multiplefunctional groups. Ligand coatings are advantageous in that more compactQDs are obtained; however, polymer coated QDs tend to have superiorbrightness and photostability. Standard bioconjugate techniques, such ascarbodiimide coupling, are widely used with both ligand and polymerstabilized QDs. Ligand coatings have also frequently enabled thepreparation of bioconjugates through self-assembly driven bycoordination to the QD surface. Quantum dots herein include those havinga simple core with or without a coat, as well as optionally coatedcore/shell QDs.

Description

As detailed herein, QDs can function in a simultaneous role as acceptorsand donors within time-gated FRET relays. Aspects are described in Algaret al., J. Am. Chem. Soc. (2012) 134: 1876-1891, incorporated herein byreference along with its Supplemental Material.

The use of a two-step FRET relay serves to encode information havingboth a spectral and temporal dimensionality. This is achieved byassembling a configuration with an initial donor luminophore that has anexcited state lifetime much longer (>10 μs) than both the intermediaryacceptor/donor and terminal acceptor luminophores in the relay (<100ns). Further, the two FRET steps are designed to be coupled but functionapproximately independently and resolvable from one another.

The technique described herein provides two novel attributes: (1) TheFRET pathway from the QD to the fluorescent dye (termed FRET₂), whichwould normally only be observable within a time-gated window ˜0-250 nsafter optical excitation, can be shifted and extended into anobservation window of at least 55-1055 μs, via the FRET pathway from thelong lifetime FRET donor to the QD (termed FRET₁). Significantly longertimescales should be achievable in view of the Tb excited state lifetimeof greater than one millisecond. (2) Spectral measurement of the FRETrelay configuration in both the 0-250 ns and 55-1055 μs observationwindows after excitation enables resolution of the FRET₁ and FRET₂processes.

In the embodiment illustrated schematically in FIG. 1, a centralCdSe/ZnS QD (˜50 ns lifetime) serves as a scaffold for the co-assemblyof initial donors and terminal acceptors, and also as the intermediaryacceptor/donor to create a FRET relay. The QDs are made water solubleusing a dithiolated poly(ethylene glycol) ligand that binds the QD. Theinitial donor is selected to be a luminescent Tb³⁺ complex (Tb) (˜2.5 mslifetime), while the terminal acceptor is selected to be Alexa Fluor 647(A647), a fluorescent dye (˜1 ns lifetime). The FRET relay assembly maybe interrogated using a commercial fluorescence plate reader capable offlash/pulsed excitation and spectral acquisition with time-gating.

Within the exemplary FRET relay, the QD served as an intermediateacceptor/donor, where: (1) an excited-state Tb donor transferred energyto the ground-state QD following a suitable microsecond delay; and (2)the QD subsequently transferred that energy to an A647 acceptor. Aphotophysical analysis was undertaken for each step of the FRET relay.The assembly of increasing ratios of Tb per QD was found to linearlyincrease the magnitude of the FRET-sensitized time-gated QDphotoluminescence intensity. The Tb was found to sensitize thesubsequent QD-A647 donor-acceptor FRET-pair without significantlyaffecting the intrinsic energy transfer efficiency within the secondstep in the relay. The utility of incorporating QDs into this type oftime-gated energy transfer configuration was demonstrated inprototypical bioassays for monitoring protease activity and nucleic acidhybridization; the latter included a dual target format where eachorthogonal FRET step transduced a separate binding event. Potentialbenefits of this time-gated FRET approach include: eliminatingbackground fluorescence, accessing two approximately independent FRETmechanisms in a single QD-bioconjugate, and multiplexed biosensing basedon spectrotemporal resolution of QD-FRET without requiring multiplecolors of QD.

The luminophores (FRET partners) used in this technique may be varied asdesired. In addition to the commercially available Lumi4®-Tb (Lumiphore,Richmond, Calif., USA) used in the examples, other long lifetime FRETdonors could be used including various luminescent lanthanide complexes(chelates or cryptates) based on Tb³⁺, Eu³⁺, Sm³⁺, Tm³⁺, and the like.Ruthenium complexes may also exhibit a sufficiently long excited statelifetime to serve as long lifetime FRET donors. Similarly, CyS, Atto647,and many other fluorescent dyes or any other short-lifetime fluorophore(e.g. fluorescent proteins) may take the place of the Alexa Fluor 647(Invitrogen by Life Technologies, Carlsbad, Calif., USA) employed in theexamples. Different QD intermediates, including CdTe/ZnS and InP/ZnS QDs(etc.) can be used as well as the CdSe/ZnS QDs used in the examples(Invitrogen by Life Technologies).

Methodology

Reagents.

CdSe/ZnS QDs were obtained from Invitrogen by Life Technologies(Carlsbad, Calif.) and functionalized with dihydrolipoic acid-appendedpoly(ethylene glycol) (PEG, MW˜750) ligands (see refs. 24 and 25).Peptides were synthesized as described in refs. 26 and 27 and labeledusing A647 maleimide (Invitrogen) or Lumi4® Tb³⁺ N-hydroxysuccinimide(NHS) complex (Tb; Lumiphore, Richmond, Calif.) (see ref. 28). Probe andcomplementary target oligonucleotides were obtained from Integrated DNATechnologies (Coralville, Iowa). Targets were labeled with A647 or Tb;probes were modified to hexahistidine (His₆)-DNA-peptide chimeras asdescribed in ref. 27. Peptide and oligonucleotide sequences are given inTable 1, and the labeling chemistry is shown in FIG. 8

TABLE 1Peptide and oligonucleotide sequences. Amino acid residues are capitalized innormal font; nucleotides are given in lower case italics.Peptides (written N- to C-terminal): PEP A¹(A647)-CSTRIDEANQRATKLP₇SH₆ (SEQ. ID No: 1) PEP B² (LUMi4™Tb³⁺-GSGAAAGLSH₆ (SEQ. ID No: 2) Oligonucleotides (PRB = probe, TGT =target): PRB A³3′-tta gtt ctg tta taa caa- 5′-CGSGAAAGLSH₆ (SEQ. ID Nos: 3 and 4) TGT A5′-aat caa gac aat att gtt- 3′-(A647) (SEQ. ID No: 5) PRB B³3′-caa cat cct aat tga ctt- 5′-CGSGAAAGLSH₆ (SEQ. ID Nos: 6 and 4)TGT B² 5′-gtt gta gga tta act gaa- 3′-(Lumi4™ Tb³⁺) (SEQ. ID No: 7)¹Peptide labeled at the cysteine (thiol). ²Peptide labeled at theN-terminus (1° amine), oligonucleotide labeled at a 3′-amino linker.³Peptide-oligonucleotide chimeras are linked by a disulfide bridgebetween the peptide cysteine residue and 5′-thiol linker on theoligonucleotide.

Instrumentation and Photoluminescence (PL) Measurements.

PL spectra were acquired using a Tecan Infinite M1000 Dual MonochromatorMultifunction Plate Reader equipped with a xenon flash lamp (Tecan,Research Triangle Park, N.C.). Non-gated PL emission spectra: 400 Hzflash frequency, 400 nm excitation, ˜0 μs delay between flash and dataacquisition, and a 40 μs integration time. Time-gated PL emissionspectra: 100 Hz flash frequency, 339 nm excitation, 55 μs delay, 1 msintegration time. Tb/QD PL decay time measurements were acquired usingthree different systems (PLD systems 1-3), each with optimizedcapabilities for different decay.

PLD system 1 measurements were made using a customized spectrometersystem with a 355 nm Nd:YAG laser source (60 Hz, 5 ns pulse width; 1 μJper pulse) and monochromator for emission wavelength selection, analogPMT signals were processed using a digital oscilloscope and the timeresolution was 40 μs, and a 55 μs time-gate was used.

PLD system 2 measurements were made using a modified KRYPTOR platereader (Cezanne, Nîmes, France) system with 4000 detection bins at 2 μsintegration steps over 8 ms. A nitrogen laser was used for excitation(337.1 nm, 20 Hz, ˜100 μJ per pulse). To detect Tb PL, a 494±20 nmbandpass filter was used; to detect Tb-sensitized QD PL, a 640±14 nmbandpass filter was used.

PLD system 3 measurements used a PicoQuant FT 300 fluorescencespectrometer, with samples at 40 nM concentration in borate buffer, pH8.5, with a 1 MHz repetition rate and emission collected at 620±5 nm.The QD PL decay lifetimes were measured with excitation at both 330 nmand 405 nm (which are close to the wavelengths used for excitation intime-gated and non-gated PL spectrum measurements). The values reportedare the intensity averaged PL lifetime from a triexponential fit.

QD Bioconjugates and Assays.

A647/Tb labeled peptides/oligonucleotides were conjugated to QDs viapolyhistidine self-assembly by mixing at the desired stoichiometricratios, in buffer, for 30-60 min. No purification was necessary, and thewell characterized, high-affinity binding resulted in nearlyquantitative assembly to the QDs (see refs. 22 and 29-31). Quantitationusing the Tb/A647 absorption of the labeled peptides provided knowledgeof the number of donors/acceptors per QD. For characterizationexperiments, the QD conjugate concentration was 45 nM (5 pmol).Time-gated proteolytic assays were done by preparing (PEPB-Tb)₁₀-QD-(PEP A-A647)₃ conjugates, adding trypsin, and trackingtime-gated PL at 625 and 675 nm over 1.5 h at 2 min intervals. The finalQD conjugate concentration was 0.2 μM (20 pmol). Time-gatedhybridization assays were done by mixing TGT A-A647 (0-50 pmol) with 60pmol of PRB A, hybridized 60 min, (PEP B-Tb)₁₀-QD conjugates added, andtime-gated PL spectrum measured after 60 min. The final QD conjugateconcentration was 45 nM (5 pmol). Two-plex hybridization assays weredone similarly, except that TGT A (0-50 pmol) and TGT B (0-80 pmol) weremixed with 50 pmol of PRB A and 80 pmol of PRB B prior to addition ofunconjugated QDs. Both non-gated and time-gated PL spectra weremeasured.

Results

Peptides and peptide-oligonucleotide chimeras used were engineered todisplay terminal His₆ metal-affinity sequences to provide spontaneousself-assembly to the Zn²⁺-rich QD surface. As noted in refs. 22, 29, and30, this motif has been used to prepare QD bioconjugates of proteins,peptides, and oligonucleotides with excellent control over the conjugatevalence. Characterization of His₆ self-assembly to QDs confirmed that anaverage of 50±10 peptides can be assembled around a ˜6 nm diameterDHLA-capped QD. The 625 nm emitting QDs used here were coated with asimilar ligand but were ˜10±1 nm in diameter, suggesting access to aneven wider range of conjugate valences. Since these experiments requiredthe assembly of peptides and oligonucleotides, the latter werechemically ligated with a His₆-appended peptide as described in ref. 29(FIG. 8 and Table 1) to ensure a level of control that was analogous topeptide assembly. In turn, labeling the peptides or oligonucleotideswith Tb and/or A647 (FIG. 8) enabled excellent control over the numberof Tb and A647 assembled per QD; this permitted characterization of theFRET₁ and FRET₂ processes (depicted in FIG. 1A) during stepwise changesin donor-acceptor stoichiometry. The schematic constructs in FIG. 1Bsummarize the three different QD bioconjugates used here: (i) peptides,(ii) peptide-oligonucleotide chimeras, and (iii) hybrid assemblies ofpeptides and peptide-oligonucleotide chimeras. Since assembly of theselabeled materials to the QD yields an approximately centrosymmetric FRETconfiguration, it was possible to treat all of the Tb or A647 in a givenassembly as being equivalent within the Förster formalism.

Donor-Acceptor Pairs and Spectral Overlap.

Red-emitting 625 nm PL QDs (27 nm full-width-at-half-maximum) werepaired as both an acceptor for the initial Tb donor, and a subsequentdonor for the A647 acceptor. FIG. 2 shows the absorption and PL spectrafor the Tb, QD and A647, as well as the spectral overlap functions forthe Tb-QD and QD-A647 FRET pairs. FIG. 2A shows absorbance and PLspectra for the Tb³⁺ complex (as PEP B-Tb), red-emitting CdSe/ZnS QDs,and A647 dye (as PEP A-A647); 339 and 400 nm excitation are shown forreference. FIG. 2B shows spectral overlap functions for the Tb-QD andQD-A647 FRET pairs. Photophysical and FRET parameters for the differentluminophores and donor-acceptor combinations are listed in Table 2. TheTb³⁺ complex incorporates a proprietary isophthalamide-type ligand thatsensitizes the lanthanide ion, which otherwise has a prohibitively lowdirect molar absorptivity, as noted in refs. 28 and 33. The Tb wasoptimally excited at 339 nm and exhibited sharp emission lines at ˜490,550, 585 and 620 nm. The QD was efficiently excited at 339 nm(ε_(QD)≈1.9×10⁷ M⁻¹ cm⁻¹) and 400 nm (ε_(QD)≈1.1×10⁷ M⁻¹ cm⁻¹). Incontrast, the A647 was only very weakly excited at these wavelengths(ε₆₄₇≦1×10⁴ M⁻¹cm⁻¹).

TABLE 2 Optical characteristics of Tb, QD and A647 luminophores withtheir FRET pairs. Luminophore ε_(max) (M⁻¹ cm⁻¹) [λ_(max)] ε(M⁻¹ cm⁻¹)[λ_(D)]^(a) Φ τ Tb  26 000 [339 nm] — 0.77 ± 0.10 2.6 ± 0.2 ms (Lumi4ligand) (Tb³⁺) QD 5.5 × 10⁵ [610^(b) nm] 5-50 × 10⁵ 0.55 ± 10    50 ± 3ns [475-575 nm] A647 239 000 [650 nm] 89 000 [625 nm] 0.33^(c) ~1 ns^(d)FRET pair (D→A)^(e) J (mol⁻¹ cm⁶) R₀ (nm) Tb → QD 7.2 × 10⁻⁹ 10.1 QD →A647 1.8 × 10⁻⁹ 7.5 FRET pairs (D→A)^(e) r_(pred.) (nm)^(f) r_(meas.)(nm) FRET modality PEPB-Tb → QD^(g) 6.2-6.7 6.3 FRET₁ (Tb lifetimequenching) QD → PEPA-A647^(h) 7.7-8.2 8.4 FRET₂ (QD PL quenching) QD →PEPA-A647^(i) 7.7-8.2 8.3 FRET₂ (QD PL quenching) non-gated with(PEPB-Tb)₁₀ QD → PEPA-A647^(j) 7.7-8.2 8.1 FRET₂ (QD PL quenching)time-gated with (PEPB-Tb)₁₀ ^(a)Extinction coefficient at peak donor PLemission wavelength, λ_(D). ^(b)Extinction coefficient at first excitonpeak. ^(c)Source: Invitrogen by Life Technologies. ^(d)Source: ref.⁵⁵^(e)Written as donor to acceptor. ^(f)Geometric prediction based on QDand peptide dimensions. ^(g)Tb → QD measured from Tb PL decay quenching.^(h)QD → A647 measured from QD PL quenching following direct QDexcitation. ^(i)QD → A647 measured from non-gated QD PL quenchingfollowing direct QD excitation (PEP B-Tb also present on QD). ^(j)QD →A647 measured from time-gated QD PL quenching following FRET₁sensitization.

The large Förster distance calculated for Tb-to-QD energy transfer (10.1nm) was a product of the extremely strong absorption of the QD acrossthe emission range of the first three Tb lines. The Förster distance ofthe QD-A647 FRET pair was 7.5 nm, and this value is among the largestnoted when pairing a QD donor with a dye acceptor (typically, R₀<6 nm).Examining the Tb-QD-A647 three-luminophore system a priori confirmed thepotential for a multistep FRET₁+FRET₂ relay process whereinexcited-state Tb can transfer energy to the QD (acceptor), whichsubsequently acts as a donor for the A647, resulting in a net energytransfer from the Tb to the A647. The putative Tb-A647 FRET pair hadsignificant spectral overlap and a Förster distance of 5.7 nm(J=2.5×10⁻¹⁰ mol⁻¹ cm⁶); however, the FRET₁ and FRET₂ pathways should bemore favored since, based on the relative Förster distances, their ratesare expected to be 30-fold and 5-fold faster than Tb-to-A647 energytransfer, respectively.

Intensity-Based Analysis of Tb-to-QD Energy Transfer (FRET₁).

Initial experiments focused on determining the degree to which the Tbcould sensitize time-gated QD PL via FRET₁. Increasing ratios ofTb-labeled PEP B (PEP B-Tb) were assembled on the 625 nm QDs and theresulting PL spectra were collected in both non-gated (˜0 μs delay) andtime-gated modes (see Materials and Methods section for exactdefinitions). Time-gating for the measurements was empirically selectedto be 55 μs, which corresponded to the minimum delay after flashexcitation needed to minimize signal from direct excitation of the QDs.The residual signal was due to an instrumental/electronic echo effectrather than residual PL, as the QD excited-state completely decayed inless than a microsecond. An integration time of 1 ms was selected fortime-gated measurements to be commensurate with the typicalexcited-state lifetimes of Tb complexes (see ref. 33). Withouttime-gating, the Tb sensitization of QD PL could not be observed overthe directly excited QD PL.

As shown in FIG. 3A, an approximately linear increase in the time-gated,Tb-sensitized QD PL was observed as the valence of PEP B-Tb assembledper QD was incrementally increased from 0 to 20 Tb per QD. An analogousexperiment using the same ratios of prehybridized Tb-labeled TGT B/PRB Bpeptide-DNA chimeras (PRB B/TGT B-Tb) is shown in FIG. 3B, and alsorevealed an approximately linear increase in QD sensitization. Increasedtime-gated QD sensitization was also observed beyond 20 Tb per QD, butthe linear trend was not always consistent. In the case of the PRB B/TGTB-Tb loading, non-specific QD adsorption of TGT was found to benegligible. The long excited-state lifetime of the Tb providedsufficient time for the QD to relax to its ground state (following flashexcitation) and function as an effective FRET acceptor. In turn, thetime-gating provided a mechanism to monitor this process. The time-gatedQD PL signal was minimal in the absence of assembled Tb, confirming thatthe His₆-mediated selective attachment of the Tb-labeledpeptides/oligonucleotides to the QD, and thereby sensitized thetime-gated QD PL.

The Tb functioned as an effective FRET donor for the QD irrespective ofwhether it was directly labeled onto a peptide terminus (PEP B-Tb) orindirectly through oligonucleotide hybridization (PRB B/TGT B-Tb). Theslightly lower rate of QD sensitization or FRET efficiency for thelatter is believed to arise from a slightly longer Tb-QD separation inthe DNA incorporating configuration. The Tb was attached at the end of asix-carbon aminated linker, which can allow some freedom of movement, inaddition to breathing of the oligonucleotide hybrids. Moreover, previousresults suggest that dye labels assembled onto QDs using similarpeptide-DNA chimeras can have a wide range of movement relative to theQD (see ref. 34). Regardless, assembling greater numbers of Tb aroundthe central QD increased the rate of energy transfer from Tb donors toQD acceptors, and this effect was observed as increases in thetime-gated QD PL sensitization.

To further assess FRET₁, the excitation spectra of (PEP B-Tb)₁₀-QDconjugates were measured with and without time-gating, as shown in FIG.3C. In both cases, the Tb emission (monitored at 490 nm), gave rise tothe characteristic PEP B-Tb excitation/absorption peak centered at 339nm. In contrast, monitoring the QD PL (at 625 nm) produced three verydifferent results. The characteristically broad QD excitation/absorptionspectrum was observed without time-gating for QD alone or (PEPB-Tb)₁₀-QD conjugates. With time-gating, the QD alone yielded no signal,whereas (PEP B-Tb)₁₀-QD conjugates exhibited the characteristic Tbexcitation/absorption peak centered at 339 nm. The latter indicated thatenergy absorbed by the Tb was being reemitted by the QD. These resultsprovided additional confirmation of QD sensitization by the Tb once theQD had returned to its ground state following direct optical excitation.It should be noted that marked quenching of the Tb donor PLintensity—especially at higher assembly ratios—could not be consistentlyobserved in the time-gated PL spectra. This result did not allow formeasurement of FRET efficiency directly from the donor PL loss and is incontrast to previous formats where QD donors were assembled with anincreasing number of acceptors, resulting in progressive quenching ofthe QD PL (see refs. 20 and 35-38). However, given the multipledonor-single acceptor configuration, this behavior was not unexpected.

PL Decay Analysis of Tb-to-QD Energy Transfer (FRET₁).

Further characterization and confirmation of Tb-to-QD energy transferwas obtained using PL decay time analyses. Measurements of the Tb PLlifetimes were first collected, using PLD system 1, as the number of PEPB-Tb assembled per QD was increased. As shown in FIGS. 4A-B, the Tblifetimes were monitored at both the 490 and 550 nm emission lines. Inthe absence of QD, the PEP B-Tb had a monoexponential PL decay with acharacteristic lifetime of ca. 2.6-2.7 ms. When an average of ˜1 PEPB-Tb was assembled per QD, the PL decay became distinctlymultiexponential, showing a fast decay component and a residuallong-lifetime component that paralleled the native Tb lifetime. As theaverage number of PEP B-Tb was increased to 2.5, 5, 10, 15 and 20 perQD, the relative contribution of the native, long-lifetime componentincreased significantly; however, the fast decay component did not fullydisappear. At >10 PEP B-Tb per QD, the ratio of the fast and nativedecay components saturated to a constant value. This behavior wasreflected in both the 490 and 550 nm Tb PL lines, although for the 550nm line, the fast decay appeared more attenuated compared to the native,long-lifetime component of the Tb. This disparity coincided with therelative brightness of the Tb lines (550 nm>490 nm; see FIG. 2A.

The appearance of the very fast decay component was consistent with veryefficient energy transfer from the Tb to QD, as expected based on thelarge R₀ of 10.1 nm for this FRET pair. It was estimated that the PEPB-Tb places the Tb≦1.2 nm from the QD surface and ˜6.7 nm from the QDcenter. This value was arrived at by considering: (1) a negligiblecontribution from the His₆-terminus which is in direct contact with theZnS shell, (2) the Ala₁ tract forming a helix that is disrupted by theflanking glycine residues, (3) rotational flexibility in the peptide,and (4) comparison to donor-acceptor distances for similarly sizedpeptides determined previously (see ref. 38). The QD radius wasestimated to be 5.5 nm. As the Tb donor-QD acceptor separation (r≈6.7nm) was much shorter than the Förster distance (10.1 nm), a FRETefficiency exceeding 92% was expected. The short Tb lifetime componentwas between ca. 20-200 μs and suggested FRET₁ efficiencies of 93-99%.However, these short lifetimes were comparable to the temporalresolution (40 μs) of PLD system 1 measurements; further experimentswere done at higher resolution (2 μs bins) using PLD system 2.Representative data measured for different ratios of Tb per QD (490 nmemission line) are shown in FIGS. 4C-D and summarized in Table 3. Theshort, QD-quenched, Tb decay component(s) were analyzed and yielded anaverage lifetime of 150±60 μs, which corresponded to a FRET₁ efficiencyof approximately 94±3%. Based on this data, the rate of FRET₁ isestimated to be 6.3×10³ s⁻¹.

TABLE 3 FRET₁ efficiencies determined from FRET₁-quenched Tb PL decaylifetimes and FRET₁-sensitized time-gated QD PL decay lifetimes(collected with PLD system 2). Tb PL (490 nm) QD PL Tb per QD_(Tb-FRET)τ_(av) (μs) τ₃ = τ_(Tb) (ms) E_(Tb) ^(a) _(QD-FRET)τ_(av) (μs)E_(QD) ^(b) 0 — 2.72 — 0.05^(c) 0 0.5 159 2.72 0.95 133 0.95 1 172 2.730.95 119 0.96 2 136 2.74 0.94 114 0.96 5 126 2.72 0.94 113 0.96^(a)FRET₁ efficiency from fast Tb PL decay component. ^(b)FRET₁efficiency from time-gated sensitized QD PL decay. ^(c)QD in the absenceof FRET; measured with PLD system 3 (see Supporting Information).

Due to electronic saturation of PLD system 2 at short time-scales, someresidual fast decay component appeared in the PEP B-Tb decay curves (seeFIG. 4D). This was a result of the high sensitivity of PLD system 2 andits optimization for long-lifetime measurements. Although thiscontribution was very small, it was important to compare the lifetimeresults from the Tb donor decays with the Tb-sensitized QD PL decays.The latter are proof of FRET since microsecond to millisecond QD PLdecay can only result from FRET-sensitization. The QDs had a native PLlifetime of ca. 50 ns following direct optical excitation (see FIG. 4E),however, with assembly of PEB B-Tb, the QD manifested a 120±30 μs PLlifetime that was significantly increased (2400-fold) and commensuratewith the fast Tb PL decay component. FIG. 4F shows plots of theTb-sensitized, QD acceptor PL decays at the same ratios of PEP B-Tb usedin FIGS. 4C-4D. This result was conclusive evidence of the FRET₁ pathwayand corresponded to an efficiency of 95±3%, which was in good agreementwith that estimated from the Tb PL decay. Assuming a FRET₁ efficiencybetween 91-98%, the Tb-QD center-to-center separation distance in (PEPB-Tb)_(n)-QD conjugates was calculated to be between 5.3-6.9 nm (6.3 nmfor the median 94.5% efficiency). This value was also in good agreementwith the estimated length of PEP B plus the QD radius (˜6.7 nm), despitethe intrinsic insensitivity of FRET to changes in donor-acceptorseparation distance at very high efficiencies.

QD-to-A647 Energy Transfer (FRET₂).

The second QD-to-A647 energy transfer (FRET₂) step in the FRET relay wasexamined. Increasing ratios of A647-labeled PEP A (PEP A-A647) wereself-assembled around the central QD, and the non-gated and time-gatedPL emission spectra measured. As shown in FIG. 5A, increasing the ratioof PEP A-A647 assembled per QD resulted in the progressive quenching ofQD PL and sensitization of A647 PL via FRET₂ in the non-gated PLspectrum. Analogous measurements of equivalent amounts of PEP A-A647without QD revealed negligible directly excited acceptor emission,confirming efficient FRET and significant reemission by the acceptor.The observed trends of increasing donor/acceptor quenching/sensitizationwere analogous to those observed with other QD donor-multiple dyeacceptor FRET pairs (see refs. 20 and 35-38). The time-gated PL spectrumof these conjugates revealed only background noise and no traces of QDor A647 PL. Fitting the non-gated FRET data with the Förster modelyielded an average donor-acceptor separation of r≈8.4 nm for the QD-(PEPA-A647)_(m) conjugates. This value agreed with predictions. In additionto a QD radius of ˜5.5 nm, PEP A comprised a Prop motif that forms atype-II helix ˜1.2 nm in length (see ref. 39) and 15 additional residuesthat contribute 1-1.5 nm of length. The overall separation was thusr≈7.7-8.2 nm. The maleimido linker in the dye structure will alsocontribute some extra length. Based on an average r≈8.3 nm (vide infra)and an intrinsic QD lifetime of 50 ns, the rate of FRET₂ was estimatedat 1.1×10⁷ s⁻¹ per acceptor. This rate corresponds to ca. 36% FRETefficiency for the first A647 acceptor.

Tb-to-QD-to-A647 Time-Gated FRET Relay (FRET₁+FRET₂).

For the next FRET characterization, PEP B-Tb and PEP A-A647 wereco-assembled around the central QD to yield the final Tb-to-QD-to-A647energy transfer relay. Time-gated PL measurements were again criticalfor observing Tb-sensitization of the QD during FRET₁, and thesubsequent energy transfer from the QD to the A647 in FRET₂. To allowsimple resolution of the effect of FRET₁ on FRET₂, the PEP B-Tb ratiowas fixed at an intermediate value of 10 per QD. This valencecorresponded to a significant rate of QD sensitization (see FIG. 3)while still leaving a large amount of the QD surface available forassembling PEP A-A647, which was added at ratios between 0-6 per QD. Asshown in the inset of FIG. 5B, the non-gated PL emission spectrum of thefull conjugate revealed the QD PL quenching and sensitization of A647 PLcharacteristic of directly excited FRET₂ and similar to that of QD withonly A647 shown in FIG. 5A. It can be seen that the data in the FIGS. 5Aand 5B insets are nearly superimposable and analysis yielded anestimated QD-A647 separation of r≈8.3 nm in the (PEP B-Tb)₁₀-QD-(PEPA-A647), conjugates without time gating—a value that deviated less than2% from that measured for the non-gated QD-(PEP A-A647)_(m) conjugates(see Table 2). Coassembly of ˜10 PEP B-Tb on the central QD thus hadlittle effect on the directly excited FRET₂ pathway.

FIG. 5B shows the time-gated PL emission spectra of the (PEPB-Tb)₁₀-QD-(PEP A-A647)_(m) conjugates. In addition to Tb PL, both QDand A647 PL were observed. Even with sensitization from the FRET₁pathway rather than direct optical excitation, the QD PL showed the sameprogressive quenching with an increasing ratio of PEP A-A647 acceptorper QD. Similarly, the A647 showed a corresponding pattern ofFRET₂-sensitized PL that increased with its valence. Notably, the Tb PLwas not significantly quenched by the addition of PEP A-A647,underlining the approximate independence of FRET₁ and FRET₂. Analysis ofthe time-gated QD PL quenching derived an average QD-A647 separation ofr≈8.1 nm—a less than 3% deviation from the other two data sets (seeTable 2). These two results confirmed that the intrinsic properties ofFRET₂ were carried over into the time-gated measurements sensitized byFRET₁. All three data sets are quantitatively compared in FIG. 5C; theymatch extremely well and can all be fit to the Förster formalism. Theaverage QD-A647 separation across the three data sets was r≈8.3 nm.Moreover, separate analysis of the FRET efficiency at each A647 valenceacross all three QD-to-A647 data sets yielded, on average, a relativestandard deviation <10%. In addition, the A647/QD acceptor/donor PLratio was also determined and compared between the same data sets. Agood correspondence is seen across the different PEP A-A647 valences,except for small negative deviations in the time-gated data at 5-6 PEPA-A647 per QD. The latter is believed to arise from poorer instrumentalsignal-to-noise (S/N) at the A647 wavelengths within the time-gatedmeasurement settings, rather than a modification of FRET₂ (similarly,S/N is poorer for Tb in non-gated measurements). This data alsosuggested that there was no significant “extra” sensitization of theA647 via direct Tb-to-A647 FRET, in agreement with our a prioriexpectations based on relative energy transfer rates.

To further establish the FRET₁+FFRET₂ relay, excitation spectra werecollected with PEP A-A647 and (PEP B-Tb)₁₀-QD-(PEP A-A647)₄ conjugates,as shown in FIG. 5D. The PEP A-A647 valence was fixed at 4 per QD toensure efficient but non-saturated FRET. The excitation spectra werecollected at 675 nm, corresponding to A647 PL emission. PEP A-A647 alonewas characterized by its own excitation/absorption profile withouttime-gating and gave rise to no measureable excitation spectrum withtime-gating. In contrast, the (PEP B-Tb)₁₀-QD-(PEP A-A647)₄ conjugateexcitation spectrum was a composite of the A647 and QDexcitation/absorption profiles without time-gating. This was indicativeof direct excitation and FRET₂, respectively. Importantly, withtime-gating, the excitation spectra corresponded to that of the Tb,unequivocally demonstrating that the time-gated sensitization of theA647 originated from the Tb via consecutive FRET₁ and FRET₂ processes atthe QD. Cumulatively, the data collected to this point also suggestedthat FRET₁ and FRET₂ were approximately independent of one another(time-gated sensitization notwithstanding).

Proteolytic Assays.

Protease sensing using the FRET relay assembly was investigated in atime-gated, kinetic mode to examine possibilities of this technique inbiosensing. This utilized PEP A-A647 and PEP B-Tb along with trypsin—aprototypical serine protease that cleaves on the C-terminal side ofarginine and lysine residues. To enable sensing, PEP A incorporated onelysine (K) and two arginine (R) cleavage sites along its length (seeref. 40). In contrast, PEP B contained no lysine or arginine residues,and was therefore not a potential substrate for trypsin (confirmedexperimentally; data not shown). The time-gated QD-FRET relay monitoredtrypsin activity by following the loss of FRET₂ from proteolysis of PEPA-A647. Analogous to previous QD-FRET configurations for sensingproteolytic activity (see refs. 36, 38, and 40) the initial state ofthis time-gated configuration was “ON” with respect to the QD-A647 FRET₂pair, as illustrated in FIG. 1B(i). Proteolysis decreased the number ofA647 proximal to the QD, progressively shifting the system toward aFRET₂ “OFF” state with increasing activity, and thus provided a dynamicsignal. In parallel, PEP B-Tb provided approximately constant time-gatedsensitization of the QD by FRET₁. (PEP B-Tb)₁₀-QD-(PEP A-A647)₃conjugates were selected since 10 equivalents of PEP B-Tb providedsignificant time-gated QD sensitization, and 3 initial equivalents ofPEP A-A647 afforded maximal changes in FRET efficiency during subsequentproteolysis. In contrast to previous configurations, time-gated sensingcould be accomplished due to the FRET₁ pathway. Another novel featurewas the measurement of protease activity in a kinetic mode, where thecourse of proteolysis was followed in real-time using two-colorratiometric measurements.

As shown in FIG. 6A, exposing the FRET-relay protease sensor toincreasing amounts of trypsin increased the rate at whichFRET-sensitized QD PL recovered, and the rate at which FRET₂-sensitizedA647 PL was lost. Accordingly, the time-dependent FRET₂ efficiencyshowed a commensurate decrease with the progression of proteolysis. Theinitial rate of change in the FRET₂ efficiency also increased linearlywith increases in protease concentration. Control experiments with notrypsin showed consistent QD PL, A647 PL, and FRET efficiency over alltime courses. For the unoptimized combination of QD-peptide substrateconcentrations, trypsin preparation, sample conditions, and analysistime utilized herein, a limit of detection (LOD) was estimated to be 200pM (0.5 ng) trypsin (using a threshold value of three standard-errorsbeyond the slope of the line of best fit through the FRET efficiencytime course for the negative control at 0 nM trypsin). This LODrepresents an approximately 3-fold and 30-fold improvement compared tothe 625 pM or 6.25 nM previously estimated for a similar QD-FRET sensorassembled using the same peptide substrate (with smaller QDs and adifferent acceptor dye), but measured in a non-kinetic mode on afluorescent plate reader or a custom microchip platform, respectively(see refs. 40 and 41). The improvement in LOD was surprising, given thatthe previous studies used QD-FRET sensors based on a single QD-to-dyeFRET pathway. However, in this FRET relay, the efficiency of FRET₁ wasvery high (˜94%) which minimized the loss of final acceptorsensitization due to the added energy transfer step. The increase insensitivity can be attributed at least in part to the added kineticanalysis, which allows greater resolution of low activity proteolysis.

Time-Gated DNA Hybridization Assay.

A second sensing configuration explored using the FRET relay was atime-gated hybridization assay. To this end, QDs coassembled with PEPB-Tb and PRB A were employed. Initial measurements were made withouttime-gating by mixing QDs with 15 equivalents of PRB A that had beenprehybridized with increasing amounts of TGT A-A647, as a QD-(PRBA)₁₅/(TGT A-A647), configuration. As shown in FIG. 6B(i), the result wasthe expected rise approaching maximum FRET efficiency, and anapproximately linear increase in the FRET-sensitized A647/QD PL ratio.The latter provided a more convenient (linear and no reference stateneeded) and sensitive capacity for quantitative detection. For thetime-gated hybridization assay, (PEP B-Tb)₁₀-QD conjugates wereco-assembled with 12 equivalents of PRB A to detect an increasingquantity of TGT A-A647, as shown in FIG. 1B(iii). Analogous to thetime-gated protease construct, the role of PEP B-Tb was to providetime-gated sensitization of the QD PL in the final (PEP B-Tb)₁₀-QD-(PRBA)₁₂/(TGT A-A647), configuration. The latter time-gated PL spectrumrevealed the expected FRET “ON” progression as the amount of hybridizedTGT A-A647 increased, indicated by decreases in QD PL and correspondingincreases in sensitized A647 PL. Between the non-gated and time-gatedformats, the FRET₂ efficiency as a function of the number of equivalentsof PEP A-A647 did not change (see FIG. 6B); however, the slope of theA647/QD PL ratio diminished, attributable to lower signal-to-noise forthe A647 PL within the time-gated measurements. Quantitative time-gateddata (FIG. 6B(ii)) was obtained from the linear increase in A647/QD PLratio, and the LOD was estimated to be 16 nM (1.8 pmol). The LODthreshold, determined at ca. 670 nm, was set as three standarddeviations above the average baseline QD PL spectrum in the region660-775 nm. That is, the minimum amount of TGT A-A647 needed to have areliably measurable A647 PL signal above the noise expected due to theQD crosstalk at ca. 670 nm, and with which to calculate an A647/QD PLratio. A continuation of the linear trend in A647/QD PL ratio was notedat a 25% excess of TGT A-A647 over PRB A, suggesting that probe-targethybridization was less than 1:1. In terms of concentration, the 16 nMLOD was approximately an order of magnitude higher than the ˜1 nM LODspreviously reported for ensemble solution-phase and solid-phasehybridization assays (see refs. 42 and 43, respectively) based on QD-dyeFRET pairs (no relay). Those assays used 500 and 1250 μL sample volumes(cf. 100 μL used herein), such that the LOD in terms of the absolutequantity of material was comparable (˜0.5-1.3 pmol). However, as aratiometric measurement, it should be noted that this value is afunction of both the QD-bioconjugate concentration and sensitivity ofthe instrumentation. In our experiments, the limitation appeared to bethe microplate reader, which was primarily designed for high-throughputanalysis instead of high sensitivity spectrofluorimetry, and preventedthe use of lower quantities of QD to detect smaller amounts of target.Nevertheless, these results confirmed that DNA hybridization could alsobe monitored using time-gated Tb-to-QD-to-A647 FRET.

Orthogonal Two-Plex DNA Hybridization Assay.

Another sensing configuration focused on exploiting the approximatelyindependent FRET₁-FRET₂ mechanisms for signal transduction in amultiplexed format. It is apparent that the two different energypathways could be increasingly sensitized by the assembly of more Tb orA647 per QD. In contrast to previous QD-based biosensing formats (see,e.g., ref. 44), the technique described herein provides a route tomultiplexed detection that does not derive its information from the useof multiple QD colors, but rather from the temporal resolution of theFRET₁ and FRET₂ processes. It was desired to demonstrate that each FRETprocess could reflect a distinct biorecognition event and provide anorthogonal analytical signal.

The magnitude of FRET₁-sensitized time-gated QD PL is linearlyproportional to the amount of proximal Tb (see FIG. 3). Likewise, theA647/QD PL ratio can be linearly proportional to the amount of proximalA647 (see FIG. 5B). For a two-plex assay, the non-gated A647/QD PL ratiowould reflect FRET₂ uniquely since the Tb signal is excluded from thesemeasurements. Thus, any biomolecular binding event associating A647 withthe QD could be detected orthogonally to any events associating the Tbwith QD. The time-gated QD PL sensitization could be used to measure theextent of FRET₁ if the quenching effect of proximal A647 is accountedfor. Thus, the total time-gated QD+A647 PL sensitization was used as ananalytical signal for the extent of the FRET₁ process. A correctionbased on the A647 quantum yield was introduced to account for energythat was transferred from the QD, but not reemitted by A647. Thisanalysis provided a working model and two predicted orthogonalanalytical signals to verify experimentally. A DNA hybridization assaywas selected since Watson-Crick base-pairing is a selectivebiorecognition event that can be readily designed to avoidcross-reactivity.

In conjunction with non-gated and time-gated measurements, a two-plexconfiguration was created by assembling (PRB B)₁₆-QD-(PRB A)₁₀conjugates to respond to TGT B-Tb and TGT A-A647. The oligonucleotideswere prehybridized with target, the QDs added, and self-assembly allowedto occur yielding (TGT B-Tb)_(n)/(PRB B)₁₆-QD-(PRB A)₁₀/(TGT A-A647)_(m)conjugates for detection. FIG. 1B(ii) illustrates the genericbioconjugate structure, while the schematics in FIG. 7A depict thedifferent permutations of target hybridization-mediated FRET within thebioconjugates at the four extremes of the assay: m=n=0; m=10, n=0; m=0,n=16; and m=10, n=16. The first permutation (i) corresponded to anabsence of target and only the QD, which gave rise to QD PL withouttime-gating, and no signal with time-gating. Permutation (ii)corresponded to the hybridization of TGT A-A647, which resulted in amixture of QD and FRET₂-sensitized A647 PL in non-gated measurements,and no signal in time-gated measurements. The third permutation (iii)corresponded to the hybridization of TGT B-Tb, which yielded only QD PLwithout time-gating, and a mixture of Tb and FRET₁-sensitized QD PL withtime-gating. Permutation (iv) corresponded to hybridization of both TGTA-A647 and TGT B-Tb. Here, the non-gated spectrum showed QD andFRET₂-sensitized A647 PL, whereas the time-gated spectrum showed Tb PL,FRET₁-sensitized QD PL and FRET₂-sensitized A647 PL, reflecting assemblyof the full Tb-to-QD-to-A647 FRET relay.

The orthogonality of the two-plex hybridization assay was evaluatedusing an array of different mixtures of TGT A-A647 and TGT B-Tb,followed by calculation of the non-gated A647/QD PL ratio (FRET₂) andtime-gated total QD+A647 PL sensitization (FRET₁) from the measured PLspectrum of each mixture. As the amount of TGT A-A647 increased, thenon-gated A647/QD PL ratio increased linearly, but was relativelyunaffected by the presence or absence of TGT B-Tb (FIG. 7B(i)).Similarly, as the amount of TGT B-Tb increased, the total QD+A647 PLsensitization increased linearly, but was minimally affected by changesin the presence of TGT A-A647 (FIG. 7B(ii)). The data in FIG. 7B(i-ii)were collected simultaneously in the same experiment and from the samesamples. These results effectively demonstrated orthogonal quantitativeresponses, where the A647/QD PL ratio responded to TGT A-A647, and thetotal QD+A647 PL sensitization responded to TGT B-Tb. For a rigorousdemonstration of the concept and analysis, the small amount ofoverlapping Tb PL were numerically deconvolved from the QD PL in themeasured two-plex PL spectra (alternative analyses are discussed later).The LOD for TGT A-A647 and TGT B-Tb were estimated to be 17 nM (1.9pmol) and 29 nM (3.2 pmol), respectively. The LOD thresholds were takenas three standard deviations above the average A647/QD PL ratio (0 nMTGT A-A647, 0-727 nM TGT B-Tb, time-gated) or total QD+A647 PLsensitization (0 nM TGT B-Tb, 0-454 nM TGT A-A647, non-gated). Despitethe two-plex format, these LODs compare favorably to the time-gated,one-plex hybridization assay.

Due to the emission rate of the long lifetime FRET donor, the FRET₁pathway is poorly observed promptly after interrogation (<100 ns), butreadily observed over extended observation times (10⁵-10⁶ ns). This aidsdirectly in distinguishing between the two FRET steps. The number andproximity of long lifetime FRET donors and fluorescent dyes assembled tothe QD determines the rate of FRET₁ and FRET₂, respectively. Controlover these parameters thus enables the modulation of FRET₁ and FRET₂,thereby permitting spectral-temporal encoding of information.

Applications

Potential applications areas utilizing luminescent probes or reportersin non-biological or biological applications requiring, or benefitingfrom, extending the observation window of FRET pair. Other applicationsinclude labeling, assays, or chemo/biosensing on the surface of orwithin cells, tissues (in vitro or in vivo), environmental samples,and/or other complex sample matrices prone to high levels of opticalsource scattering and/or autofluorescence that can be ameliorated bytime-gating.

Potential applications also exist in areas where luminescent probes orreporters in biological applications requiring, or benefiting from,multiplexed detection in a spectro-temporal format. This includes:labeling, assays, or chemo/biosensing in vitro or within cells andtissues.

Further potential application areas are those where luminescence can beused for unique identification, tracking, or validation/authentication.This includes: optical barcodes for commercial/shipping use, and/oranti-counterfeit measures/forgery deterrent.

This technique may also be used advatageously in areas where traditionalFRET has been employed, including measurements of binding and/ordissociation, enzymatic activity, protein folding, and cellularprocesses (such as endocytosis and protein synthesis).

Nanoparticles such as QDs also show great promise for a new generationof diagnostic probes, biomedical technologies, therapeutics, andsingle-vector combinations thereof, sometimes called “theranostics.”

Advantages

QDs as Intermediate Acceptors/Donors in FRET.

Several properties of QDs are uniquely advantageous for assembling thetwo-step energy transfer relay, particularly when the QD is configuredas an intermediary. The strong, broad QD absorption was resonant withthe three strongest Tb emission lines and was characterized byextinction coefficients approaching an order of magnitude greater thanthat of most fluorescent dye acceptors (see ref. 45). The use of afluorescent dye intermediary in the FRET relay configuration would alsobe hindered by a narrower absorption band that would only be resonantwith two of the Tb emission lines. As a consequence, the QD acceptoroffered a much larger spectral overlap integral and Förster distancewith the Tb. The QD PL was also well separated from the Tb PL (exceptfor the Tb emission line at 620 nm, which was the least intense of allthe lines). In turn, when the QD functioned as the donor for the finalA647 acceptor in the relay, the narrow QD PL provided strong overlapwith the A647 absorption, while its peak remained spectrallywell-resolved from the A647 emission at 675 nm. The latter would nothave been possible with a fluorescent dye intermediary, given thecharacteristic broad and red-tailed dye emission profile.

The non-trivial surface area of the QD was of almost equal importance toits optical properties, and its utilization as a central nanoscaffoldgreatly facilitated the physical assembly of the energy transfer relay.In contrast, the use of a fluorescent dye intermediary in the relaywould require an extrinsic scaffold, such as a protein ordouble-stranded DNA, to provide the necessary proximity with the initialdonor(s) and final acceptor(s). Such a scaffold would not readilyprovide the approximately centrosymmetric distribution of Tb and A647that was achieved with the QD intermediary, and which enabledstraightforward analysis. Further, since the QD was its own intrinsicscaffold, it enabled the use of peptides and nucleic acids in abiological rather than structural motif. A maximum of 20 total peptides(PEP A+PEP B) were assembled per QD here, which is less than half of themaximum packing expected for even smaller QDs. This “extra” surfaceavailability could allow the assembly of one or more other biomaterialsto the QD in order to provide utility beyond the FRET relay. Forexample, cell penetrating or other targeting peptides could be added toinduce cellular uptake or in vivo targeting of the final bioconjugate asdescribed in refs. 46-51.

The properties of His₆ self-assembly for binding to the QD also providedadvantages. Utilizing the His₆ motif offered simplicity, efficiency, andreproducibility. QD bioconjugation required only mixing of the QD andthe desired equivalents of biomolecule(s). This level of control overQD-bioconjugate valence effectively permitted tuning of the relativeefficiency of the FRET₁ and FRET₂ pathways, regardless of the use ofTb/A647 labeled His₆-appended peptides, or His₆-appended oligonucleotideprobes hybridized with labeled target. Such versatile and incrementaltuning of two distinct FRET pathways—solely through assembly valence—maybe unique to the use of QDs and His₆-bioconjugation in these types ofassemblies. In contrast, assembling biotinylated DNA tostreptavidin-coated QDs results in a large amount of heterogeneity inseparation distance, hindering control and characterization of FRET, asnoted in ref. 34. Purely synthetic chemical approaches to derivingsimilar architectures based on, for example, a dendrimeric substrate,are extremely labor intensive, and neither spin coating norlayer-by-layer approaches would provide the same levels of controland/or precision as His₆ (see refs. 17 and 52).

Time-Gated Biosensing.

With few exceptions, multistep FRET relays incorporatingQD-bioconjugates have been based on QD-to-dye-to-dye energy transferconfigurations (see refs. 20-22). The role of the relay in theseinstances was primarily to extend the range of energy transfer and/or toallow for wavelength shifting. Herein, the former consideration wasaddressed by the large Förster distances of the FRET₁ (10.1 nm) andFRET₂ (7.5 nm) pairs, while the latter was addressed by pairing ared-emitting QD with the deeper-red A647 acceptor. The unique featuresof the Tb-to-QD-to-A647 FRET relay were the added ability to maketime-gated QD PL measurements, and sensitization of QD-to-A647 energytransfer over a millisecond timescale. With time-gating, undesirabledirect excitation of the final A647 acceptor—regardless of theexcitation wavelength used—was avoided due to its ˜1 ns fluorescencelifetime. However, a potentially more important advantage arises fromthe <20 ns characteristic decay times of cellular and tissueautofluorescence (see ref. 56), such that the time-gating afforded byTb-to-QD FRET is expected to permit the separation of analytical PLsignals from unwanted background PL in almost any complex biologicalmatrix. While the protease sensing demonstrated in this work wasprimarily proof-of-concept for demonstrating utility of the FRET relay,it should be noted that abnormal protease activity is associated withmany diseases, including ischemia, autoimmune and neurodegenerativedisease, as well as several types of cancers (see ref. 57). Thisrelevance suggests, for example, the possibility of sensitive,time-gated, in situ measurements of protease activity associated withover-expressed extracellular matrix metalloproteinases in complex tumormilieu. Use in complex biological matrices will be predicated on thefidelity of the QD-conjugate assemblies therein. Complexation of the Tbis highly stable and selective for lanthanide ions, polyhistidine is notan endogenous motif, and PEG coatings are largely biocompatible—all ofwhich suggest that preassembled QD-conjugates should remain functionalin biological matrices.

In addition to the advantages of time-gating via the Tb-QD FRET pair,there are advantages associated with the ratiometric detection affordedby the QD-A647 FRET pair. Ratiometric analyses tend to be relativelyinsensitive to dilutions and small variations in excitation intensity orbetween different instruments. This format is also particularly wellsuited to assays in a kinetic format, where donor and acceptor PLintensities dynamically change over extended time periods (e.g. hours)and are highly susceptible to instrumental drift and noise.

Orthogonal Spectrotemporal Multiplexing.

In previous FRET sensing configurations based on QD donors, multiplexedinformation has been encoded by using two different colors of QD witheither a common or different FRET acceptors (refs. 60 and 35,respectively). Multiplexing configurations where QDs have been used asFRET acceptors also rely on multiple QD colors (refs. 11 and 14). Inpractice, ratiometric methods based on use of different fluorescentacceptor(s) have been preferred, and the number of colors measured (i.e.wavelength bands) was always greater than the number of targets.Moreover, multiplexing in these formats was usually limited to theensemble since each individual QD-bioconjugate still only transduced onetype of target. Compared to this state-of-the-art, the Tb-to-QD-to-A647FRET relay offers an important and unique possibility in multiplexedbioanalysis: the ability to simultaneously transduce the activity of twodifferent biomolecular targets by using a single color of QD incombination with orthogonal PL signals that are sensitive to differentluminophores (the latter are bound to the QDs via biomolecularrecognition). Furthermore, since these signals were proportional to thenumber of proximal acceptors, the viability of this assay format wasdemonstrated in a semi-quantitative, two-plex hybridization assay (FIG.7).

The use of full PL spectra and numerical deconvolution to resolveoverlapping Tb and QD PL was performed for characterization andvalidation of the two-plex strategy, and is not generally expected to berequired in the majority of practical applications of the FRET relayassembly. The small degree of Tb crosstalk in measuring QD PL onlyappeared in the time-gated measurement and systematically added to theapparent magnitude of the latter, such that it was readily calibratedinto quantitative results. Alternatively, the use of 605 nm PL QDs,which would also be a suitable intermediary for the Tb and A647 in arelay, could potentially avoid this limited crosstalk since its PLmaximum falls between the 585 and 620 nm Tb emission lines. In eithercase, the Tb PL intensity need not factor into the analysis, therebypermitting the use of two-color detection centered on the QD and A647PL. Importantly, this two-color advantage does not come at the cost ofratiometric measurements. Since the A647 and QD PL manifests in both thenon-gated and time-gated PL spectra, the FRET₁ pathway can be measuredrelative to the directly excited FRET₂ pathway by dividing thetime-gated A647+QD PL sensitization (FRET₁ signal) by the non-gatedA647+QD PL, which is a reference state independent of FRET₁. Thus, bothFRET₁ and FRET₂ can be analyzed ratiometrically. The caveat of thisFRET-relay strategy is that only static biological processes, or thosewith slow dynamics (≧10⁻² s), can be monitored due to the need formicrosecond time-gating and a millisecond integration time.

The orthogonal calibration curves in FIG. 7B are a powerful confirmationof the approximate independence of the FRET₁ and FRET₂ pathways, as wellas the strong biosensing potential of this time-gated, multiplexed FRETconfiguration. Dependence on directly-labeled targets can be avoided byusing labeled reporter oligonucleotides in a sandwich format (refs. 43,61, and 62), intercalating dyes (refs. 35, 42, and 63), or molecularbeacon configurations (refs. 29,64, and 65). Simultaneous monitoring oftwo different proteases should also be feasible in this spectrotemporalformat, as well as targeting of many other enzymatic processes (refs. 14and 66). An intriguing idea is utilizing the FRET₁ and FRET₂ processesto monitor and correlate physically-associated, coupled, or cascadedevents while still using a single QD assembly. For example, certainproteases have differential sensitivity to phosphorylated versusnon-phosphorylated peptide substrates. This capability could allow thecorrelation of kinase/phosphatase activity that is coupled to subsequentproteolysis. Beyond in vitro applicability, access to multiplexedsensing using a single compact QD-based probe can reduce the amount ofextraneous material that must be delivered intracellularly. This featurepotentially lessens the perturbation of a cellular system under study,and/or avoids challenges associated with differences in the cellulardelivery efficiency between two distinct probes. It would also reducesome optical complexity in multiplexed microscopy systems (e.g. fourcolor channels for two distinct donor-acceptor pairs without a relay,vs. two color channels and electronics for time-gating when utilizingsuch a relay).

The advantages of the techniques described herein include the following:

1. The use of a long-lifetime (microsecond to millisecond) initial donorfor FRET₁ enables time-gated observation, with good fidelity, of an(approximately) independent FRET₂ process (intermediary-terminalacceptor pair), which would otherwise only be visible with promptobservation. Time-gated observation allows for the collection andaccumulation of large amounts of data, with improved signal-to-noisewith weak signals, and can be useful in situations when multiple eventsmight contribute to the same signal.

2. The time-gated observation window that is suitable for theamelioration of scattered source light and sample/matrixautofluorescence such as that found in biological samples and in vivocellular and tissue formats.

3. The FRET₂ pair can be spectrally observed at their characteristicwavelengths in both prompt and time-gated observation after pulse/flashexcitation.

4. The two-step FRET relay is a discrete entity (compared with multipleone-step FRET pairs).

5. Approximately independent tuning of FRET₁ and FRET₂ by controllingthe number of initial donors and terminal acceptors, respectively, inassociation with the intermediary acceptor/donor.

6. Approximately independent tuning of FRET₁ and FRET₂ by controllingthe proximity between the intermediary acceptor/donor and the initialdonor(s) and terminal acceptor(s), respectively.

7. Three-fold flexibility in tuning the FRET₁ and FRET₂ efficiencies bythe selection of the initial donor, intermediary acceptor/donor, and/orterminal acceptor on the basis of spectral overlap.

8. The two-step FRET relay requires less spectral bandwidth and/orchannels to encode, for example, two-plex information when compared totwo discrete FRET pairs.

9. Spectro-temporal information is more difficult to replicate thansolely spectral information (e.g. as an authenticator/forgerydeterrent).

10. The two-step FRET relay can potentially be used in solution, at aninterface, within cellular environments, or embedded within matricessuch as host nanoparticles or polymeric films/coatings.

11. Applicability and compatibility with both biological andnon-biological environments.

12. Beyond the long lifetime FRET donor- or dye-labeled peptide andoligonucleotide assembly to QDs demonstrated herein, two-step FRETrelays can be comprised of fluorescent dyes and long lifetime FRETdonors labeled/conjugated to a variety of other biomolecules, organic,or inorganic scaffolds that assemble to, or incorporate, a suitableintermediary acceptor/donor.

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CONCLUDING REMARKS

All documents mentioned herein are hereby incorporated by reference forthe purpose of disclosing and describing the particular materials andmethodologies for which the document was cited.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

What is claimed is:
 1. A method of using a FRET relay assembly, themethod comprising: providing a FRET relay assembly comprising: a longlifetime FRET donor, a semiconductor quantum dot (QD) configured as anintermediate acceptor/donor in FRET and assembled to the long lifetimeFRET donor in sufficient proximity thereto so as to allow for a firstFRET process between the long lifetime FRET donor and the QD and afluorescent dye configured as a terminal FRET acceptor and assembled tothe QD in sufficient proximity thereto so as to allow for a second FRETprocess between the QD and the fluorescent dye, wherein the longlifetime FRET donor has an excited state lifetime of at least onemicrosecond and the QD and fluorescent dye each have excited statelifetimes of less than 100 nanoseconds; spectrally exciting the FRETrelay assembly; and receiving a spectral emission from the FRET relayassembly.
 2. The method of claim 1, wherein at least two distinct FRETrelay assemblies are provided.
 3. The method of claim 1, whereinspectral emissions are received in observation windows 0-250 ns and55-1055 μs after said spectrally exciting.
 4. The method of claim 1,wherein said long lifetime FRET donor comprises Tb³⁺, Eu³⁺, Sm³⁺, Tm³⁺,or Ru²⁺.
 5. The method of claim 1, wherein said long lifetime FRET donorand/or said fluorescent dye are bound to said QD using peptides and/oroligonucleotides.
 6. The method of claim 1, wherein at least one His₆motif is adapted to bind said long lifetime FRET donor and/or saidfluorescent dye to said QD.
 7. The method of claim 1, wherein the QD isfunctionalized with carboxylate, amine, or poly(ethylene glycol).